RESUMO
Migraine patients are particularly prone to develop medication overuse headache (MOH). However, the risk factors for the transformation of migraine to MOH are still not clear. We investigated gene polymorphisms, personality traits, and characteristics of headache and lifestyle in 47 migraine patients (aged 36.4 ± 10.3) and 22 MOH patients (aged 39.6 ± 9.9) who progressed from migraine and made a scoring system for a predictive index (PI) of the onset of MOH in patients with migraine. By multivariate logistic stepwise regression analysis, type of migraine, regular and sufficient dietary intake, and methylenetetrahydrofolate reductase (MTHFR) C677T (rs1801133) and dopamine D2 receptor (DRD2) C939T (rs6275) polymorphisms were selected as significant factors that contribute independently to the development from migraine to MOH (P < 0.05). The regression coefficients (ß) of these four selected factors were approximated and scored. The PI score in MOH patients (7.32 ± 1.60) was significantly higher than that in migraine patients (4.62 ± 1.83, P < 0.001). The proposed scoring system should in the future be the object of larger studies to confirm its validity.
Assuntos
Transtornos da Cefaleia Secundários/epidemiologia , Transtornos de Enxaqueca/complicações , Adulto , Idade de Início , Primers do DNA , Feminino , Genótipo , Transtornos da Cefaleia Secundários/genética , Transtornos da Cefaleia Secundários/psicologia , Humanos , Estilo de Vida , Modelos Logísticos , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/genética , Enxaqueca com Aura/complicações , Enxaqueca com Aura/tratamento farmacológico , Enxaqueca sem Aura/complicações , Enxaqueca sem Aura/tratamento farmacológico , Personalidade , Testes de Personalidade , Polimorfismo Genético/genética , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Dopamina D2/genética , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
Parvalbumin (PA), one of the Ca2+-binding neuronal marker proteins, has been revealed to exist in the myelinated axons of the posterior root of the spinal cord and the peripheral nerve of rats. To investigate the role of PA for the genesis of diabetic neuropathy, the levels of PA in the sciatic nerve of normal and streptozotocin-induced diabetic rats were measured by radioimmunoassay (RIA) for PA. The immunohistochemical distribution of PA in the sciatic nerve from both groups was also studied. The RIA for PA revealed that the levels of PA in the sciatic nerve of diabetic rats were significantly decreased when compared with those of normal rats. However, the contents of S-100 protein, another type of Ca2+-binding glial marker protein, did not show any significant difference in the sciatic nerve from both groups. Immunohistochemically, the amount of PA containing myelinated axons of the diabetic nerve was markedly decreased when compared with nondiabetic subjects. These results suggest that the decreased level of PA in the peripheral nerve might contribute to the genesis of diabetic neuropathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Proteínas Musculares/metabolismo , Parvalbuminas/metabolismo , Nervo Isquiático/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Masculino , Radioimunoensaio , Ratos , Ratos Endogâmicos , Proteínas S100/metabolismo , Nervo Isquiático/patologiaRESUMO
Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue.
Assuntos
Carcinoma Papilar/metabolismo , Proteínas de Transporte/metabolismo , Iodetos/metabolismo , Proteínas de Membrana/metabolismo , Sódio/metabolismo , Simportadores , Neoplasias da Glândula Tireoide/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunológicas , Iodo/metabolismo , RNA Mensageiro/genética , RNA Neoplásico/genética , Glândula Tireoide/metabolismo , Células Tumorais Cultivadas/metabolismoRESUMO
Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes.
Assuntos
GMP Cíclico/biossíntese , Glucose/farmacologia , Óxido Nítrico/farmacologia , Sistema Nervoso Periférico/metabolismo , Sequência de Bases , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/metabolismo , Neuropatias Diabéticas/etiologia , Glucose/análogos & derivados , Humanos , Inositol/farmacologia , Modelos Neurológicos , Dados de Sequência Molecular , Neuroblastoma , Óxido Nítrico Sintase/antagonistas & inibidores , Sistema Nervoso Periférico/efeitos dos fármacos , Sistema Nervoso Periférico/enzimologia , Proteína Quinase C/metabolismo , Sorbitol/metabolismo , Células Tumorais CultivadasRESUMO
Gene therapy with thyroglobulin (TG) promoter and a prodrug/suicide gene combination may prove useful as a treatment for thyroid carcinoma. However, most poorly differentiated and anaplastic thyroid carcinomas have lost the ability to express the TG gene expression accompanied by loss of transcription factors [thyroid transcription factor-1 (TTF-1), TTF-2, or Pax-8] interacting with the TG promoter. In anticipation of developing transcriptionally targeted gene therapy of TG-nonproducing thyroid carcinomas, we investigated the effect of TTF-1 gene transfer on TG promoter activity and the cytotoxic effect obtained by the TG promoter-driven HSV-TK gene along with ganciclovir in thyroid carcinoma and nonthyroidal cells. Using a chimeric construct containing the 5'-flanking region of the rat TG gene between -826 and +39 bp and the luciferase gene, TG promoter activity was detected in a normal rat thyroid cell line (FRTL-5), but not in a dedifferentiated line of thyroid cells (FRT) expressing Pax-8 but not TTF-1, TTF-2, or TG [TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)], or in a human papillary thyroid carcinoma cell line [BHP15-3; TTF-1(-)/TTF-2(-)/Pax-8(-)/TG(-)], a human pulmonary cell line [H441; TTF-1(+)/TTF-2(-)/Pax-8(-)/TG(-)], or a dog kidney epithelial cell line [MDCK; TTF-1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. Cotransfection of the TTF-1 expression vector stimulated TG promoter activity in FRT and BHP15-3 dedifferentiated thyroid cells, but not in H441 pulmonary cells. Only weak activation was observed in MDCK kidney cells. We then constructed recombinant adenovirus vectors, AdTTF-1 and ADTGTK: AdTTF-1 contained cytomegalovirus promoter and rat TTF-1 cDNA; AdTGTK carried the TG promoter-driven HSV-TK gene. Infection with AdTGTK and combined with GCV treatment induced a cytotoxic effect in FRTL-5 cells but not in dedifferentiated thyroid or nonthyroid cells. Cotransduction of AdTTF-1 and AdTGTK permitted 90% cytotoxicity for BHP15-3 and >95% cytotoxicity for FRT, as well as for BHP7-13 and BHP18-21v thyroid cancer cell lines [both/TTF1(-)/TTF-2(-)/Pax-8(+)/TG(-)]. In contrast, little cytotoxicity was seen for H441 and MDCK cell lines even with 300 microg/ml of ganciclovir. These results suggest that cotransduction of a TG promoter-controlled suicide gene and the TTF-1 gene by adenoviral vectors confers transcriptionally targeted gene-mediated cytotoxicity in poorly differentiated thyroid carcinoma cells unable to express the TG gene.
Assuntos
Carcinoma Papilar/genética , Terapia Genética/métodos , Proteínas Nucleares/genética , Tireoglobulina/genética , Neoplasias da Glândula Tireoide/genética , Fatores de Transcrição/genética , Ativação Transcricional , Adenoviridae/genética , Animais , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/terapia , Ganciclovir/farmacocinética , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Regiões Promotoras Genéticas , Ratos , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Tireoglobulina/biossíntese , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/terapia , Fator Nuclear 1 de Tireoide , Transdução Genética , Células Tumorais CultivadasRESUMO
Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Ciclo Celular , Transformação Celular Neoplásica , Clonagem Molecular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/imunologia , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Proteínas Serina-Treonina Quinases/imunologia , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glândula Tireoide/enzimologiaRESUMO
GLUT4, the major glucose transporter in adipose tissue, is expressed during the differentiation of 3T3-L1 cells from preadipocytes to adipocytes. We previously examined the mouse GLUT4 promoter activity up to -590 bp, and demonstrated that the 5'-flanking region of the GLUT4 gene between -200 and -100 bp contains sequences that act as a repressor in preadipocytes, but not in adipocytes. Here we examine in detail the activity of this repressor in 3T3-L1 cells. Transient transfections indicated that the region extending from -125 to -112 bp functions as a repressor element only in preadipocytes. In electrophoretic mobility shift assay (EMSA), this GLUT4 repressor element (G4RE) generated specific bands with nuclear extracts from preadipocytes, but not from adipocytes. Southwestern blot analysis identified a protein of approximately 96 kDa from preadipocytes that bound to the G4RE site. Mutation of the G4RE site, which abolished the protein/DNA complex formation by EMSA, increased GLUT4 promoter activity only in preadipocytes. These results suggest that the G4RE site and its binding protein may regulate GLUT4 gene transcription during adipocyte differentiation.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Regulação da Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Musculares , Proteínas Repressoras/metabolismo , Transcrição Gênica , Células 3T3 , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Transportador de Glucose Tipo 4 , Camundongos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , TransfecçãoRESUMO
GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation.
Assuntos
Células 3T3/citologia , Células 3T3/metabolismo , Metilação de DNA , Expressão Gênica/fisiologia , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Musculares , Adipócitos/fisiologia , Animais , Diferenciação Celular/fisiologia , Transportador de Glucose Tipo 4 , Camundongos , Regiões Promotoras Genéticas/fisiologia , Células-Tronco/fisiologia , Transcrição Gênica/fisiologiaRESUMO
From a family of 16 diabetic patients with typical maternal inheritance, we investigated a 69-year-old woman with type 2 diabetes. The proband showed no major deletions in the mitochondrial DNA (mtDNA). Direct sequencing revealed 7 missense and 5 ribosomal RNA homoplasmic nucleotide substitutions when compared with the Cambridge Sequence and its recent revision. When compared with the control cybrid cells, the proband cybrid cells showed 6 nucleotide substitutions. Among these, 14577 T/C, which turned out to be 98.9% heteroplasmic, is a new missense substitution in the NADH dehydrogenase 6 gene. We also observed 2 other patients with 14577 T/C substitution from another group of 252 unrelated diabetic patients, whereas no individual from a group of 529 control subjects had 14577 T/C substitution. Furthermore, these 6 substitutions were in linkage disequilibrium. Mitochondrial respiratory chain complex I activity and O2 consumption rates of the proband cybrid cells, which were obtained by the fusion of mtDNA-deleted (rho0) HeLa cells and mtDNA from the proband, showed 64.5 and 61.5% reductions, respectively, compared with control cybrid cells. The present study strongly indicates that the new mtDNA mutation at 14577 T/C is probably a major pathogenic mutation for type 2 diabetes in this family.
Assuntos
DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Impressão Genômica , Mutação de Sentido Incorreto , NADH Desidrogenase/genética , Idoso , Grupo dos Citocromos b/genética , Diabetes Mellitus Tipo 2/enzimologia , Feminino , Células HeLa , Humanos , Células Híbridas , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Linhagem , RNA Ribossômico/genética , RNA Ribossômico 16S/genéticaRESUMO
Platelet function in patients with NIDDM is enhanced. We have found that spontaneous aggregation (i.e., the formation of small-sized aggregates in the absence of agonist stimulation) occurs at a high rate in platelets from NIDDM patients. We then investigated basal myosin light chain 20 (MLC) phosphorylation, which plays a key role in platelet shape change and aggregation, using a monoclonal antibody against a phosphorylation site (serine 19 residue) in the MLC molecule in platelets from these patients. Standard calibration curves obtained from purified MLC or the phosphorylated form of myosin light chain 20 (MLC-P) were linear within the range of 0-150 ng for MLC and 0-3 ng for MLC-P. The amount of MLC or MLC-P in platelets was estimated, and basal MLC phosphorylation was calculated. Platelets were obtained from 9 young healthy control subjects, 13 age- and sex-matched nondiabetic control subjects, and 13 patients with NIDDM. The basal MLC phosphorylation in platelets was significantly higher in the NIDDM patients than in the control subjects, irrespective of age. These findings suggest that platelets from NIDDM patients are activated in vivo. Platelets obtained from NIDDM patients generated spontaneous aggregation, the degree of which was significantly higher than that in control subjects. Platelet spontaneous aggregation correlated well with basal MLC phosphorylation. These findings suggest that increases in basal MLC in platelets may be one factor leading to hyperaggregability of platelets in these patients.
Assuntos
Plaquetas/fisiologia , Diabetes Mellitus Tipo 2/sangue , Cadeias Leves de Miosina/sangue , Agregação Plaquetária , Adulto , Anticorpos Monoclonais , Plaquetas/metabolismo , Colesterol/sangue , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fosforilação , Valores de Referência , Triglicerídeos/sangueRESUMO
The stimulation of iodide (I-) transport by TSH in FRTL-5 thyroid cells is partly due to an increase in Na+/I- symporter (NIS) gene expression. The identification of a TSH-responsive element (TRE) in the NIS promoter and its relationship to the action of thyroid transcription factor-1 (TTF-1) on the promoter are the subjects of this report. By transfecting NIS promoter-luciferase chimeric plasmids into FRTL-5 cells in the presence or absence of TSH, we identify a TRE between -420 and -370 bp of the NIS 5'-flanking region. Nuclear extracts from FRTL-5 cells cultured in the absence of TSH form two groups of protein-DNA complexes, A and B, in gel mobility shift assays using an oligonucleotide having the sequence from -420 to -385 bp. Only the A complex is increased by exposure of FRTL-5 cells to TSH or forskolin. The addition of TSH to FRTL-5 cells can increase the A complex at 3-6 h, reaching a maximum at 12 h. FRTL-5, but not nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cell nuclear extracts, form the A complex. The TSH-increased nuclear factor in FRTL-5 cells interacting with the NIS TRE is distinct from TTF-1, thyroid transcription factor-2, or Pax-8, as evidenced by the absence of competition using oligonucleotides specific for these factors in gel shift assays. Neither is it the nuclear protein interacting with cAMP response element. The TRE is in the upstream of a TTF-1-binding site, -245 to -230 bp. Mutation of the TRE causing a loss of TSH responsiveness also decreases TTF-1-induced promoter activity in a transfection experiment. The formation of the A complex between FRTL-5 nuclear extracts and the NIS TRE is redox-regulated. In sum, TSH/cAMP-induced up-regulation of the NIS requires a novel thyroid transcription factor, which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Nucleares/fisiologia , Simportadores , Glândula Tireoide/fisiologia , Tireotropina/farmacologia , Fatores de Transcrição/fisiologia , Regulação para Cima/genética , Animais , Proteínas de Transporte/efeitos dos fármacos , Linhagem Celular , Metilação de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica/efeitos dos fármacos , Iodo/fisiologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas Nucleares/genética , Oxirredução , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos , Ratos Endogâmicos BUF , Proteínas Repressoras/genética , Glândula Tireoide/citologia , Fator Nuclear 1 de Tireoide , Transativadores/genética , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits, a and beta1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) -116 to -80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt -116 to -80. Supershift assays with antibodies to GABPalpha and to GABPbeta1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPalpha and GABPbeta1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt -93 and -85. These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Receptores da Tireotropina/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Extratos Celulares , Ilhas de CpG , Proteínas de Ligação a DNA/genética , Fator de Transcrição de Proteínas de Ligação GA , Regulação da Expressão Gênica , Dados de Sequência Molecular , Ratos , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glândula Tireoide/citologia , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Tecido Adiposo/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ratos , Sequências Reguladoras de Ácido Nucleico , Elementos de Resposta , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
We cloned a 1.2-kilobase cDNA (C17-16) from a transformed FRTL thyroid cell library. Northern blot analysis revealed that the size of the corresponding mRNA was 2.0 kilobases. C17-16 mRNA was found in all tissues investigated, but interestingly, its expression was 5- to 10-fold higher in the thyroid glands than in other tissues. Addition of TSH to FRTL cells showed a time- and dose-dependent increase in the steady state level of C17-16 mRNA, and the effect was mimicked by (Bu)2cAMP. An in vitro nuclear run-off assay demonstrated that the stimulatory effect was due to an increase in the transcription rate of the C17-16 gene. TSH had no effect on the half-life of the C17-16 mRNA. Transcriptional induction of the C17-16 gene was inhibited when the cells were treated with cycloheximide. Nucleotide sequencing revealed that C17-16 was identical to the cDNA of rat glucose-regulated protein (GRP78), a member of the heat shock protein family. These results suggest that the expression of GRP78 in thyroid cells is regulated by TSH via cAMP, for which cycloheximide-sensitive protein synthesis might be required, and that more GRP78 might be needed to assist in the synthesis and transport of glycoprotein molecules in TSH-stimulated cells.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico , Chaperonas Moleculares , Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA/genética , Chaperona BiP do Retículo Endoplasmático , Epitélio/metabolismo , Meia-Vida , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cinética , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Distribuição Tecidual , Transcrição Gênica/efeitos dos fármacosRESUMO
We have cloned 15 kbp of rat thyroid Na+/I- symporter gene from liver genomic library, which contains 6 kbp upstream sequence from the translation initiation site. Southern blot analysis of the genomic DNA from the liver has revealed that thyroid Na+/I- symporter gene is the single gene in the rat. To study the tissue-selective expression mechanism of the gene, we at first determined the transcriptional start site of the gene. Results of a rapid amplification of cDNA end procedure as well as that of primer extension analysis indicated that the transcriptional start sites clustered between -96, -95, and -93 bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9 kbp (-1967 to -46 bp) of the 5'-flanking sequence of the Na+/I- symporter gene and luciferase gene expressed significant enzyme activity when transfected into a rat thyroid cell line, FRTL-5, but little activity was observed in BRL-3A rat liver cells. Deletion analysis of the constructs indicated that a minimal region, exhibiting promoter activity and cell specificity, is located between -291 and -134 bp of the gene. Deoxyribonuclease I footprinting shows that nuclear extracts from FRTL-5, but not BRL-3A, cells protect a region between -245 and -230 bp. Electrophoretic mobility shift assays have demonstrated that nuclear extracts from FRTL-5 cells formed a specific DNA-protein complex with an oligonucleotide probe corresponding to -250 to -211 bp of the gene, but that from BRL-3A cells did not, suggesting that thyrocyte-selective nuclear factors bind to the region. When the nuclear extracts from FRTL-5 cells were preincubated with antibody against thyroid transcription factor-1 (TTF-1), homeodomain containing nuclear protein, formation of the complex was abolished and the band was supershifted. We also found that the probe formed a DNA-protein complex with the recombinant TTF-1 homeodomain, and mutations of the binding site eliminated factor binding. When pRc/CMV-TTF-1 was cotransfected with the minimal promoter fragment of thyroid Na+/I- symporter gene into FRT cells, which express no TTF-1, it caused a significant increase in the transcriptional activity of the reporter construct, but not of the construct having mutated TTF-1-binding element. These results suggest that TTF-1 confers the cell-selective expression of Na+/I- symporter gene in thyrocytes.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Simportadores , Glândula Tireoide/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Pegada de DNA , Biblioteca Gênica , Genes Reporter , Fígado/química , Masculino , Dados de Sequência Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Fator Nuclear 1 de Tireoide , Transcrição GênicaRESUMO
The polyol pathway has been implicated in the pathogenesis of diabetic complications. To determine the activity of the polyol pathway, the ratio of erythrocyte sorbitol to blood glucose, which reflects aldose reductase activity, was evaluated in 329 patients with type II (non-insulin-dependent) diabetes mellitus and in 100 nondiabetic age-matched control subjects. Although erythrocyte sorbitol levels were markedly elevated, sorbitol-glucose ratios were significantly lower in diabetic patients than in nondiabetic subjects. Sorbitol-glucose ratios in diabetic patients decreased progressively as blood glucose and hemoglobin A1c (HbA1c) levels increased. Sorbitol-glucose ratios were also studied during a 75-g oral glucose tolerance test. Ratios were again lower in diabetic patients than those in nondiabetic subjects and significantly decreased 120 min after glucose loading. The ratio in diabetic patients also fell with increasing age of the patients. In diabetic patients with neuropathy, retinopathy, or nephropathy, however, sorbitol-glucose ratios were significantly higher than in those without these complications; ratios increased further as complications became more severe. Our findings suggest that the affinity of aldose reductase for glucose in patients with diabetic complications may be increased and that the polyol pathway is implicated in the pathogenesis of diabetic complications.
Assuntos
Biomarcadores/sangue , Glicemia/análise , Diabetes Mellitus Tipo 2/sangue , Eritrócitos/análise , Sorbitol/sangue , Neuropatias Diabéticas/sangue , Retinopatia Diabética/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valores de ReferênciaRESUMO
Simple and rapid purification procedures for parvalbumin, one of the Ca2+-binding proteins (extracted from rat skeletal muscle), were developed, and its antiserum was produced in rabbits to measure the parvalbumin content of various rat tissues by RIA. The heat treatment, ammonium sulfate fractionation, and trichloroacetic acid precipitation of soluble fraction from rat skeletal muscle followed by single diethylaminoethyl Sephadex A-50 column chromatography yielded a pure 120 mg protein from 150 g skeletal muscle. Amino acid analysis, together with electrophoretic mobility, indicated that the protein was identical to parvalbumin. The antisera to this rat skeletal muscle parvalbumin (raised in rabbits) did not cross-react with calmodulin or S-100 proteins. The RIA for parvalbumin using this antisera and [125I]parvalbumin revealed that skeletal muscle and brain contained high levels of the antigen; the values of which were 69,486 +/- 4,933.1 and 881 +/- 165.6 ng/mg protein, respectively. However, the parvalbumin antigen in the heart, lung, liver, and spleen could not be detected. On the other hand, the contents of the antigen in the endocrine glands (in nanograms per mg protein) were as follows: pituitary (125 +/- 46.6), thyroid (108 +/- 50.0), adrenal (341 +/- 64.3), testes (227 +/- 37.2), and ovaries (218 +/- 10.1). All of these values were comparable to levels of antigen found in the brain sample. These results suggest an important role for parvalbumin in endocrine glands.
Assuntos
Glândulas Endócrinas/análise , Proteínas Musculares/análise , Músculos/análise , Parvalbuminas/análise , Glândulas Suprarrenais/análise , Animais , Química Encefálica , Cálcio/metabolismo , Feminino , Imunodifusão , Masculino , Ovário/análise , Hipófise/análise , Ligação Proteica , Radioimunoensaio , Ratos , Testículo/análise , Glândula Tireoide/análiseRESUMO
We have investigated the relationship between cAMP and sodium,potassium-ATPase (Na+,K(+)-ATPase) activity in the sciatic nerves of rats treated with cilostazol, a potent phosphodiesterase inhibitor; iloprost, a stable prostacyclin analog; or (Bu)2cAMP, a cAMP analog, which increase cAMP content by different mechanisms. In in vivo studies, administration of cilostazol (20 mg/kg BW.day), iloprost (4 micrograms/kg BW.day), or (Bu)2cAMP (4 mg/kg BW.day) for 4 weeks restored decreased cAMP content and Na+,K(+)-ATPase activity in the sciatic nerves of diabetic rats and further improved motor nerve conduction velocities without alteration of myo-inositol contents. There was a positive correlation between cAMP contents and Na+,K(+)-ATPase activities in the sciatic nerves. In in vitro experiments, cAMP accumulation and Na+,K(+)-ATPase activity in the desheathed sciatic nerve blocks obtained from both normal and diabetic rats were significantly increased by incubation with cilostazol, iloprost, or (Bu)2cAMP. In addition, cAMP accumulation and Na+,K(+)-ATPase activities in endoneurial preparations incubated in both normal and high glucose buffer were also significantly increased by cilostazol, iloprost, and (Bu)2cAMP. These results strongly suggest that there is a close relationship between cAMP content and Na+,K(+)-ATPase activity in rat sciatic nerves. Therefore, cAMP content may play an important role in the development of diabetic neuropathy by modulating Na+,K(+)-ATPase activity in the peripheral nerves.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Bucladesina/farmacologia , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/metabolismo , Iloprosta/farmacologia , Nervo Isquiático/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tetrazóis/farmacologia , Animais , Cilostazol , Diabetes Mellitus Experimental/enzimologia , Técnicas In Vitro , Inositol/metabolismo , Cinética , Masculino , Bainha de Mielina/fisiologia , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Wistar , Valores de Referência , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/enzimologiaRESUMO
The possible role of the pentose phosphate shunt in thyroid hormone secretion was investigated in vivo and in vitro with mouse thyroid glands. Thyroidal endocytosis in response to TSH, a step of thyroid hormone secretion, was evaluated for its dependency upon the pentose phosphate shunt by using 6-aminonicotinamide (6-AN), an antimetabolite in the synthesis of pyridine nucleotides. Formation of 14CO2 from glucose labeled either in the C-1 or C-6 position was studied to estimate the pentose phosphate shunt activity. A dose of 6-AN markedly reduced oxidation of [1-14C]glucose but did not affect that of [6-14C]glucose induced by TSH. Concomitantly there was a marked decrease in thyroidal endocytotic response to TSH. These inhibitions by 6-AN were completely abolished by the pretreatment with nicotinamide. Methylene blue, which oxidizes NADPH and thus stimulates activity of the pentose shunt, significantly depressed thyroidal endocytosis in response to TSH in vitro. These inhibitions of colloid droplet formation by 6-AN or methylene blue were not manifested against dibutyryl cyclic AMP stimulation. Furthermore, a dose of 6-AN, which seems to inhibit only the pentose phosphate shunt, markedly depressed TSH-induced formation of cyclic amp. These findings suggest that the pentose phosphate shunt might play an important role in triggering TSH stimulation of thyroid hormone secretion by supplying NADPH, and further, that NADPH dependency in thyroid hormone secretion is at a site prior to the generation of cyclic AMP.
Assuntos
6-Aminonicotinamida , Niacinamida/análogos & derivados , Pentosefosfatos/metabolismo , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Tireotropina/farmacologia , 6-Aminonicotinamida/farmacologia , Animais , Bucladesina/farmacologia , Interações Medicamentosas , Endocitose/efeitos dos fármacos , Glucose/metabolismo , Técnicas In Vitro , Masculino , Azul de Metileno/farmacologia , Camundongos , Niacinamida/farmacologia , Prostaglandinas E/farmacologiaRESUMO
Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by TSH in mouse thyroid lobes. Pretreatment of thyroid lobes with Con A at concentrations from 1.55--400 microgram/ml was found to have biphasic stimulatory and inhibitory effects of the TSH-induced accumulation of cAMP and formation of colloid droplets. Low concentrations of Con A potentiated TSH activation of thyroidal formation of cAMP and endocytosis. In contrast, higher concentrations of Con A markedly inhibited these TSH effects. The inhibitory effects observed after preincubation with Con A were abolished by the addition of alpha-methyl-D-glucoside to the incubation medium. A high concentration of Con A also inhibited cAMP formation induced either by prostaglandin E2 or the long-acting thyroid stimulator. However, the basal and TSH-stimulated glucose oxidation in mouse thyroid lobes was not depressed by a high concentration of Con A. Uptake of 125 I-labeled Con A by thyroid tissues increased with time up to 1 h and was directly proportional to tissue weight. These findings suggest that the specific interaction between Con A and its receptors may lead to conformational changes in the structure of the membranes of the thyroid follicular cells which facilitate TSH-induced thyroid hormone secretion via the adenylate cyclase-cAMP system.